Clostridium difficile is the cause of significant morbidity, mortality and cost. Systemic and mucosal immunity to C. difficile toxin A has been associated with protection from clinical disease, amelioration of clinical symptoms, and prevention of relapse. The previous funding period of this grant resulted in several advances in the use of live, oral, attenuated Vibrio cholerae as a vector for immunizing against heterologous antigens. The long term goal of the next, proposed segment of funding is to develop and test in animals a V. cholerae-based vector vaccine that will stimulate systemic and mucosal humoral immunity against toxin A of C. difficile, and that is appropriate for ultimate human use. The current proposal has FOUR SPECIFIC AIMS to achieve this long term goal. These aims are: 1. To develop and analyze in vitro an anti-C. difficile V. cholerae-based vaccine vector. A glutamine auxotroph of V. cholerae vaccine strain CVD 103-HgR (a safe and immunogenic vaccine strain in North American human volunteer studies) will be engineered to express toxin A-HlyA (a fusion protein between a non-toxic 720 amino acid C. difficile toxin A fragment and the E. coli hemolysin A secretion signal) from plasmids of various copy numbers that complement the glutamine auxotrophy. The vaccine vector strains will also express HlyBD (the pore-forming proteins that mediate extracellular secretion of toxin A-HlyA), and an immunoadjuvant molecule, such as LT(R192G) (a non-toxic derivative of Escherichia coli heat-labile enterotoxin that retains immunoadjuvancy). Expression and cellular localization of toxin A-HlyA and LT(R192G) will be evaluated, as well as viability and stability of the vaccines in vitro. 2. The viability, stability, immunogenicity, and reactogenicity of the various oral vaccine constructs will then be evaluated in mice. 3. A combination oral priming and transcutaneous boosting immunization strategy (the latter with C. difficile toxin A toxoid with or without an immunoadjuvant) will be evaluated in mice for production of both mucosal and systemic immunity to toxin A. 4. The immunogenicity, reactogenicity, and protective efficacy of the most promising vaccine strategy will be assessed in rabbits, the latter will be measured using a challenge assay in which purified C. difficile toxin A is injected into ligated ileal loops of vaccinated and control animals.